Novel compositions and methods for prevention and treatment of protozoal disease

ABSTRACT

A composition is provided that has been specially adapted for parenteral administration, e.g., intranasal, intramuscular, subcutaneous, transdermal or intraveneous administration, wherein the composition is comprised of at least one anti-protozoal drug in a therapeutically effective amount for the treatment or prevention of protozoan infections in man an in animals. In one embodiment, the anti-protozoal drug is a triazine-based anticoccidial agent, e.g., a triazinedione or triazinetrione such as diclazuril, toltrazuril, sulfonotoltrazuril or water-soluble sodium salts thereof. In a presently preferred embodiment, the triazine-based anticoccidial agent is sulfonototrazuril. Methods of treatment of protozoal infections in man and animals are also provided.

[0001] This application claims the benefit of priority in U.S.Provisional Application Serial No. 60/103,543, filed on Oct. 8, 1998 andin U.S. Provisional Application Serial No. 60/112,175, filed on Dec. 14,1998.

FIELD OF THE INVENTION

[0002] The present invention relates to the treatment and prevention ofdiseases caused by protozoan parasites in man and in animals. Inparticular, the invention relates to novel compositions and methods forparenteral treatment and prevention of protozoal diseases, e.g.,Piroplasmosis, Babesosis, Toxoplasmosis Neospora caninum,Crytosporidiosis and Equine Protozoal Myeloencephalitis.

BACKGROUND OF THE INVENTION

[0003] Protozoan parasites (also known as apicomplexan parasites) causea variety of clinical disease manifestations in both man and animals.For example, hemoprotozoan parasites of the Babesia genus, namelyBabesia caballi and Babesia equi, are responsible for the economicallydevastating disease, equine piroplasmosis. Equine piroplasmosis iswidely distributed worldwide although it is most prevalent in thetropics, sub-tropics and temperate regions (see, Robinson, Edward N.,“Current Therapy In Equine Medicine”, Vol.2, pp.299-300, (1987) (ISBN:0-7216-1491-4)).

[0004] The principal mode of transmission of the protozoan is via a tickvector, e.g., Dermacentor nitens. Clinical manifestation of acuteinfection is characterized by depression, fever, anorexia, ictericmucous membranes, ecchymotic hemorrhages and edema of the extremitiesand ventral abdomen. Death can occur within 24-48 hours and mortalityrates in outbreaks may be high (see, Robinson, Edward, N., “CurrentTherapy in Equine Medicine”, Vol. 3, pp. 499-500 (1992) (ISBN:0-7216-3475-3)).

[0005] Horses which test positive for piroplasmosis (complement fixationor indirect flourescent antibody tests) are rarely allowed to enter theUnited States without first undergoing treatment for the disease orunder rigid guidelines (see, Brooks, L., “Piroplasmosis: The OlympicQuestion”, The Horse, pp. 43-48 (July 1996)). Currently recommendedtreatment regimens include the use of imidocarb dipropionate (BurroughsWelcome Co.) and often the treatment produces adverse side effects whichinclude salivation, restlessness, colic and gastrointestinal tracthypermotility (see, Kobluk, Calvin N. et al., “The Horse Diseases &Clinical Management”, Vol. 2, pp. 1084-1885 (1995) (ISBN: Vol.20-7216-5984-5)). Moreover, treatment with imidocarb dipropionate has metwith marginal success, especially when the etiologic agent is Babesiaequi (50% to 60%) (see, Reed, Stephen M. et al., “Equine InternalMedicine”, pp. 570-571 (1998) (ISBN: 0-7216-3524-5)).

[0006] Thus, there is a need in the art for an effective anti-protozoalagent and a method of treatment and prevention of Piroplasmosis whichdoes not produce the adverse side effects seen with current treatmentregimens.

[0007] Other examples of blood borne protozoal infection caused byBabesia spp. include: babesiosis of cattle, e.g., Babesia bigemina andBabesia bovis; caprine and ovine babesiosis, e.g., Babesia ovis; andcanine babesiosis, e.g., Babesia canis and Babesia gibsoni (see, Smith,Bradford P., “Large Animal Internal Medicine” pp.1088-1092 (1990) (ISBN:0-8016-5062-3)). See also, Bonagura, John D. “Kirk's Current VeterinaryTherapy XII Small Animal Practice”, Vol. 12, pp. 315-319, (1995) (ISBN:0-7216-5188-7)). Likewise, there is a need in the art for a safe,effective and economical treatment for such infections.

[0008] In humans, for example, protozoan infections can cause severedisease manifestations. A common sequella in patients suffering fromacquired immune defficiency syndrone (AIDS) is Cryptosporidium parvuminfection (Cryptosporidiosis) which produces severe chronic and oftenfatal diarrhea. The parasite is found worldwide and lives in cattle anddomestic animals and is excreted in feces. It can be transmitted tohumans directly from animals or through contact with feces, contaminatedwater or food (see, e.g., “National Institute of Allergy and InfectiousDiseases—AIDS-Related Cryptosporidiosis”, www press release (March,1991)).

[0009] Many attempts have been made to find a threapeutically effectivetreatment for this disease. One family of drugs currently used in theveterinary profession for the treatment of coccidosis, thetriazine-based anticoccidial agents (e.g., triazinediones andtriazinetriones) especially diclazuril and letrazuril, have been triedexperimentally in the treatment of crytosporidiosis in man (see,National Library of Medicine, AIDSDRUGS Database, DRG-0079 (Jan. 22,1998); and (National Library of Medicine, AIDSTRIALS Database, FDA-038B(Apr. 25, 1990)). These compounds are formulated for oral administrationand have met with limited success due to poor absorption. The bestresponse to such drugs has been seen in persons with the highest bloodlevels post adminsitration (see, AIDS Treatment News, No. 1 11 (Sep. 21,1991)).

[0010] To date, however, there is still no efficacious therapy forCryptosporidial infections in man (see, Health Canada, Laboratory CentreFor Disease Control: Material Safety Data Sheet - 48, Cryptosporidiumparvum, Oct. 11, 1997 @(www.hc-sc.gc.ca/hpb/lcdc/biosafty/msds/msds/48e.html (Jul. 24, 1999)).Accordingly, there still exists an urgent need in the art for a safe andeffective pareneral formulation for the treatment and prevention ofpotozoal infections such as cryptosporidiosis or babesiosis.

[0011] Equine protozoal myeloencephalitis (EPM), a central nervoussystem disease which affects equine species, is also primarily caused bya protozoan parasite, Sarcocystis neuroma also known as Sarcocystisfalcatula. The horse is not a normal host for this protozoan (the horseis not part of the normal life cycle) and is considered to be a dead endhost. The definitive host is thought to be the opossum. Equids areinfected with the S. neuroma organisms via ingestion of food or watercontaminated with feces of an infected carnivore such as the opossum(see, Robinson, Edward N., Current Therapy in Equine Medicine: Fenger,Clara A., “Equine Protozoal Myeloencephalitis”, Vol. 4, pp. 329-333(1997) (ISBN: 0-7216-2633-5)).

[0012] Recently, other protozoan parasites have been implicated as alsoplaying an etiologic role in the pathogenesis of EPM, e.g., Neosporacaninum and Toxoplasma species. Accordingly, there still exists a needin the art for an effective treatment of EPM which demonstrates broadspectrum efficacy against all protozoan parasites in the horse inclusiveof the aforementioned organisms.

[0013] The clinical signs of EPM can vary from case to case. Generally,horses present neurological signs which are asymmetrical, and actualsymptoms will vary depending upon the severity and location of lesionsproduced by the parasites in the brain, brain stem or spinal cord.Ataxia, incoordination and general weakness are usually present and canbe accompanied by muscle atrophy (usually most notable in the rearlimbs). There can be paralysis of the muscles of the eyes and face,drooping ears, difficulty swallowing (dysphagia) head tilt, alteredgait, or even seizures and collapse. Recent reports of numbers of EPMcases indicate that the disease is far more widespread and serious thanoriginally thought.

[0014] There is currently no vaccine available for prevention of thisdisease. Previously preferred treatment was aimed at control of theparasitic infection via the use of sulphonamides and pyrimethamine (see,U.S. Pat. No. 5,747,476). However, these measures have been met withlimited success. More recently, and due to the urgent need for a safeand effective treatment for this devastating disease, new methods oftherapy and new agents have been explored, e.g., through the emergencyFDA importation of anticoccidial agents such as diclazuril andtoltrazuril (see, FDACVM publication: “Instructions for Personal UseImportation of Diclazuril” (Dec. 16, 1997); and FDACVM publication: “TheImportation of Toltrazuril for Personal Use” (1997) which are availablefrom the American Association of Equine Practitioners, Lexington, Ky.(AAEP)). (See also, U.S. Pat. No. 5,883,095).

[0015] It should be noted that the current emergency importation andtreatment of horses with diclazuril and toltrazuril is speculative. Oralformulations adapted for use in the horse of one or more of these drugsare currently in clinical trials for FDA approval. And, while somehorses do dramatically improve, many only see moderate improvement(improve 1-2 grades on the clinical evaluation scale (ranging in gradesof 1-5) used by veterinarians to classify the severity of clinicalsigns). The test dose for a presently preferred form of toltrazuril, themetabolite toltrazuril sulfone (Bayer) is currently 5 mg/kg up to 10mg/kg per day for the FDA trials.

[0016] The cost for importation and use of the above anticoccidialsranges anywhere from about $800-$1,200.00 per horse and results obtainedfrom the treatment can be disheartening. Few horses experience completerecovery from any known conventional therapy, including thetriazine-based anticoccidial therapies which are mentioned above.Moreover, the relatively high levels of drug recommended in thetherapeutic regimen can produce unwanted side effects. This isespecially true for treatment regimens which utilize sulfonamides andpyrimethamine which inhibit folic acid production (see, Fenger, ClaraA., “Update on the Diagnosis of Equine Protozoal Myeloencephalitis(EPM)” Proc. 13^(th) ACVIM Forum, pp.597-600 (1995); and Bertone, JosephJ., “Update On Equine Protozoal Myeloencephalitis”, FDA Veterinarian,Vol. XI, No. III (May/June 1996)).

[0017] Therefore, there exists a need in the art to provide a safe andmore effective method of parenteral treatment of EPM which is also lesscostly.

SUMMARY OF THE INVENTION

[0018] The present invention satisfies the need in the art by providinga composition containing at least one anti-protozoal drug especiallyadapted for parenteral administration, e.g., intranasal, intramuscular,subcutaneous, transdermal or intraveneous administration, for thetreatment and prevention of protozoan infections in man an in animals.In one embodiment, the anti-protozoal drug is a triazine-basedanticoccidial agent, e.g., a triazinedione or triazinetrione such asdiclazuril or toltrazuril. In a presently preferred embodiment, thetriazine-based anticoccidial agent is sulfonotoltrazuril.

[0019] The compositions of the invention can further comprise a suitablesolvent for the anti-protozoal drug in a formulation specially adaptedfor a particular route of parenteral administration. The choice ofsolvent and concentration of active dissolved therein will of coursevary depending upon the choice of drug, the desired route of parenteraladministration, the species and host being treated, and the desiredduration of action of the administration, e.g., sustained release vs.loading dose.

[0020] Parenteral administration of the compositions of the inventionreduces the dosage amount of drug by about one fold to as much as onehundred fold but especially about five fold compared with oral dosing.The compositions provided by the invention eliminate the variability inplasma concentrations of the drug due to animal to animal differences inoral bioavailability, allow use of a loading dose and thereforeimmediate attainment of effective plasma concentrations of the drug,allow rapid attainment of high plasma concentration of the drug to drivethe drug into extravascular compartments such as the cerbrospinal fluidin the CNS, provide better and more immediate control of the plasmaconcentration of the drug and reduce the potential for side effectsrelated to the currently existing oral formulations. As a result, thecost of treatment is greatly reduced along with the potential foradverse side effects as seen from higher doses currently recommended andneeded for oral administration of these anti-protozoal drugs.

[0021] In particular, the present invention provides novel compositionsand methods for the treatment and/or prevention of any anti-protozoal orapicomplexan parasite in man and animals, e.g., equine piroplasmosis,equine protozoal myeloencephalitis and Cryptosporidiosis. In oneembodiment, the invention provides a composition comprised of atriazine-based anticoccidial drug and a suitable solvent useful for thetreatment of anti-protozoal infections. A preferred embodiment of theinvention comprises a composition comprised of diclazuril in solutionwith DMSO, DMA or mixtures thereof which is formulated for parenteraladministration for the treatment of anti-protozoal infection in man orin animals as provided by the methods of the invention.

[0022] Another preferred embodiment of the invention comprises acomposition comprised of toltrazuril, toltrazuril sulfone,sulfonotoltrazuril or mixtures thereof in solution with DMSO, DMA ormixtures thereof which is formulated for parenteral administration forthe treatment of anti-protozoal infection in man or in animals asprovided by the methods of the invention.

[0023] Yet another preferred embodiment of the invention comprises acomposition formulated for parenteral or oral administration fortreatment of anti-protozoal infections in man and in animals comprisedof a soluble salt, e.g., a sodium salt, of a triazine-basedanticoccidial agent, e.g., clazuril, diclazuril, letrazuril,toltrazuril, toltrazuril sulfone, or sulfonotoltrazuril and the like.

[0024] Also provided by the invention is a method for preparation ofwater-soluble forms of triazine-based anticoccidial agents, e.g.,clazuril, diclazuril, letrazuril, toltrazuril, toltrazuril sulfone, orsulfonotoltrazuril for use in the methods of treatment of anti-protozoalinfections provided by the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention provides a composition and method for theparenteral treatment of protozoan (apicoplexan) infections in man and inanimals. The protozoan parasite can be any protozoan known to infect manor animals, including, but not limited to, e.g., Babesia spp.Sarcocystis spp. Neosporum spp. Crytosporidium spp. Toxoplasma spp. andthe like.

[0026] It is contemplated that the compositions of the invention can beformulated for any parenteral administration. It is specificallycontemplated that intravenous, intramuscular, transdermal, intranasaland subcutaneous routes of administration can be utilized foradministration of the compositions of the invention. Specificformulations of the compositions of the invention can include powders,gels, ointments, creams, solutions, suspensions, sustained releasepreparations, patches and the like.

[0027] In one embodiment, the invention provides a composition speciallyadapted for intravenous, intramuscular, subcutaneous, or intranasaladministration which is useful for the treatment of a protozoalinfection in man or in animals, e.g., for treatment of equinepiroplasmosis, equine protozoal myeloencephalitis or humancyrptosporidiosis infection, wherein the composition comprises at leastone chemical agent which has anti-protozoal activity. The compositionsprovided herein can include any anti-protozoal agent, but especiallyanti-coccidial agents such as any of the class of triazine-basedanti-coccidial agents (i.e., agents which contain a triazine ring, e.g.,the 1, 2, 4 triazine ring or the 1, 3, 5 triazine ring configurations(see, e.g. “A 3D-QSAR Study of Anticoccidial Triazines Using MolecularShape Analysis”, J. Chem. Inf. Comput. Sci., Vol. 35, 771-778 (1995);U.S. Pat. No. 4,837,216; and U.S. Pat. No. 4,952,570, the contents ofwhich are incorporated herein by reference).

[0028] Specific examples of such agents include, but are not limited to,clazuril, diclazuril, toltrazuril, toltrazuril sulfone, orsulfonotoltrazuril. For example, the chemical structures of severaltriazine-based compounds useful in the compositions and methods setforth herein are shown below:

[0029] It can be appreciated that other anti-protozoal agents includingtheir derivatives, analogs, isomers, salts, and natural metabolites ofthese agents can also be utilized in the compositions for parenteraltreatment and prevention of any protozoal infection in man or inanimals. In a presently preferred embodiment, the triazine-basedanticoccidial agent is sulfonotoltrazuril, a toltrazuril metabolite.

[0030] Sufonotoltrazuril, the toltrazuril metabolite, contains the thiogroup of toltrazuril that has been oxidized to a sulfono group therebydiffering toltrazuril from its dioxo sulfonotoltrazuril derivative.

[0031] The chemical name used in the Chemical Abstract Database for oneform of the sulfonotoltrazuril contemplated by the invention is:

[0032]1-methyl-3-[3-methyl-4-(4-trifluoromethanesulfonyl-phenoxy)-phenyl]-[1,3,5]triazinane-2,4,6-trione.

[0033] The CAS catalog number of this compound is CAS No. 69004-04-2.The Beilstein Registry Number is 870959. The Molecular Formula isC₁₈H₁₄F₃N₃O₆S and the Molecular Mass is 457.38 g/mol.

[0034] In particular, the invention provides a composition which isuseful for parenteral treatment and/or prevention of EPM which utilizesbetween about 1/4th to about 1/100th of the amount of an anti-protozoaldrug which is necessary for oral administration in the treatment of EPM.In one embodiment of the invention, the compositions are adapted forintranasal administration and comprise about 1/10th of the currentlyrecommended dosage. In another embodiment, the compositions are adaptedfor other parenteral administration (e.g., intravenous, subcutaneous andintramuscular) and comprise between about 1/4th to about 1/100th of thecurrently recommended oral dosage, but especially about 1/3rd to about1/10th of the currently recommended oral dosage for EPM treatment. In apreferred embodiment, the intranasal and parenteral compositions of theinvention set forth above are formulated for sustained release as setforth in greater detail below.

[0035] For example, the currently recommended dosage for oral diclazuril(CLINACOX®, Pharmacea Upjohn, Canada) in the horse for an EPM treatmentregimen is about 2.5 grams of diclazuril per 1000 pound horse per day(5.5 mg/kg) administered once daily for 28 days. This amounts to about70 grams of diclazuril per horse per treatment regimen.

[0036] By contrast, the presently preferred dosage range for theparenteral compositions of the present invention for treatment of EPMwhich are comprised of similar triazine-based agents, e.g., diclazurilor toltrazuril and the like, is from between about 0.1 mg/kg to about 10mg/kg. However, the skilled artisan can appreciate that this range canvary from between about 0.01 mg/kg to about 20 mg/kg depending upon thespecific formulation, route of administration, the desired effect(loading dose vs. sustained release) and the duration of the treatmentregimen.

[0037] A presently preferred embodiment of the invention comprises acomposition adapted for parenteral administration wherein theanti-protozoal agent is selected from, but not limited to the groupconsisting of clazuril, diclazuril, letrazuril, toltrazuril, toltrazurilsulfone, and sulfonotoltrazuril or a sodium salt thereof and a suitablesolvent. The solvent can be any suitable solvent for use in animals andman and will, of course vary depending upon the choice of anti-protozoalagent and the route of administration. Presently preferred solventsinclude, but are not limited to DMSO, DMA, ethanol, water and the likeas set forth more fully below.

[0038] The preferred compositions can be utilized in methods oftreatment of anti-protozoal infections in man and in animals. Forexample, a presently preferred treatment regimen for treatment of EPMcomprises administering to a 1000 lb horse a composition for intravenousadministration comprised of between about 50 mg to about 1,500 mg, butespecially between about 250 mg and about 1000 mg and most preferablyabout 500 mg (about 1.1 mg/kg) of diclazuril, toltrazuril, toltrazurilsulfone or sulfonotoltrazuril dissolved in a suitable volume of DMSO,DMA or the like. Suitable amounts of solvent will vary from betweenabout 2 ml to about 30 ml per unit dose depending upon the choice of theanti-protozoal agent and the choice of solvent. According to the methodsset forth herein, the composition can given once per day (SID) for aduration of between about 10 and about 35 days but especially betweenabout 20 and about 30 days and most preferably about 28 days.Alternatively, a loading dose of the composition can be given to achieverapid critical plasma concentrations on day one of the regimen followedby a maintenance dose (see, Example 1 below) for a shorter duration oftherapy, e.g., between about 15 and about 25 days.

[0039] In yet another embodiment, the compositions can be speciallyformulated for parenteral use, e.g., intramuscular or subcutaneous,sustained release, e.g., a microsphere or methylcellulose preparationsuch that a single sustained release administration or a weeklyadministration of sustained a sustained release formulation of theanti-protozoal agent is possible. Alternatively, a single intraveneousloading dose followed by a sustained release intramuscular orsubcutaneous dose for maintaining sustained critical blood levels iscontemplated. In EPM, for example, the critical plasma concentration canrange from about 5 μg/ml of the antiprotozoal agent to about 12 μg/ml,but especially about 8 μg/ml.

[0040] INTRAVENEOUS ADMINISTRATION EXAMPLES

[0041] As set forth in Table 1 above, the horse MR. OWENS, (about 1,000lbs.) received 750 mg of diclazuril powder dissolved in 30 ml of DMSO,intravenously, twice on day one, at zero and zero plus one hours for atotal dose of 1,500 mg. Blood samples were drawn at the indicated timepoints immediately before the next dosing and the solid circles (

) in FIG. I represent plasma concentrations of diclazuril after theseadministrations. Plasma concentrations of dicalzuril in ng/ml arerepresented on the vertical axis against time in days on the horizontalaxis. Note the 6,000 ng/ml of diclazuril peak blood level at 24 hoursafter dosing, and the approximately 48 hour plasma half-life, consistentwith previously reported data on the plasma half-life of dicalzuril inthe horse.

[0042] The horse, DEEP POWDER, 1160 lbs., in Table 2 above wasadministered diclazuril at a dose of 0.5 mg/lb. (580 mg) in DMSO IV oncea day for eight days. Plasma levels of diclazuril at 24 hours after eachdose administration and just before the next day's dosing arerepresented by the solid circles (

) in the above Table 2. Note how the dose of 580 mg/1,0000 lbs/dayyields a stepwise increasing plasma concentrations of diclazuril,yielding a final steady state plasma concentration of diclazuril ofabout 10,000 ng/ml. Note also the close comparison between these dataand data previously presented on CLINICOX®, where daily oraladministration of about 2.5 g/1,000 lbs. of diclazuril as CLINICOX®)yielded broadly similar data. The data show that IV administration ofabout 0.5 mg/1,000 lbs. of dicalzuril in DMSO produced equivalent plasmaconcentrations of diclazuril as about five times this dose ofCLINICOX®orally, suggesting about 20% or less oral bioavailability ofthis agent.

[0043] Pharmacokinetics and the summation principle teaches us thatadministration of a loading dose such as that presented in Example 1, MROWENS, followed by a daily maintenance dose such as that presented inExample 2, DEEP POWDER, should allow rapid attainment and maintenance ofa desired or therapeutically optimal plasma concentration of (about 8-10ug/ml in plasma) diclazuril. Table 3 above shows the estimated plasmalevels that would be obtained in horses of the approximately 1,000 lb.body weights of MR. OWENS and DEEP POWDER if the 1,500 mg loading dosewas administered on day I, followed by daily maintenance doses of 500 mgday as was administered to DEEP POWDER. In this way, and following thesegeneral protocols and principles, it would be possible to rapidly attainfull effective plasma concentrations of dicalzuril or a related agent onday one of treatment and maintain these effective concentrations for anydesired period of time.

[0044] The advantages of this approach are many fold: In the firstplace, effective plasma and cerebrospinal fluid concentrations ofdicalzuril or a similar agent are attained within hours or minutes ofthe start of treatment, which can lead to shorter treatment periods. Inthe second place, the required plasma concentrations can be maintainedby the administration of only about one fifth or less of the total drugamount that must be administered orally, based on our experience withCLINICOX® administration, which may lead to substantial savings on drugcosts. Thirdly, the significant horse to horse variability inbioavailability of this agent and the resulting variability in plasmaconcentrations of diclazuril found after oral administration can beavoided, leading to more predicable and reproducible treatment results.Fourthly, using this methodology, very high plasma concentrations ofdiclazuril can be rapidly attained, leading to equivalently rapid entryof diclazuril into the CNS and to equivalently rapid anti-protozoalactions in the CNS. Fifthly, the optimal plasma concentrations ofdicalzuril to treat this disease and the optimal duration of therapy andmost cost effective therapeutic approaches to EPM have yet to beconclusively established.

[0045] This IV approach to administration of diclazuril, by allowingprecise control of drug concentrations and rapid entry of drug into theCNS, provides a tool necessary to precisely control the plasma levels ofthe drug and cerebrospinal fluid concentrations of this agent to obtainthis information. Sixth and finally, the fact that IV administrationexposes the horse to less of this drug and to none by the directintestinal route reduces the probability of adverse effects developingrelating to the gastrointestinal system and also to other body systemsof the horse.

[0046] Pharmacokintetic analysis of the data obtained after IVadministration of this drug and comparison with previously reportedpharmacokinetic data after administration of CLINICOX® (Table 4 above)shows that the estimated oral bioavailability of CLINICOX® may beactually less than the earlier estimated 20%. In Table 2, theintravenous data from MR. OWENS (solid squares

) is plotted along with the previously obtained CLINICOX®oraladministration data (solid circles

) and subjected to pharmacokinetic analysis as set forth below. Thesedata suggest that the bioavailability of orally administered CLINICOX®isabout 13% less that the figure developed in the experiments presentedabove.

Example #4

[0047] Mr. Owens/CLINACOX Data: Pharmacokinetic Analysis andBioavailability Calculations/Continued:

[0048] Following oral administration, concentration of diclazuril at 120hours was considered as outliers.

[0049] Area Under the Curve (AUC from 0 to ∞): Trapezoidal rule was usedto calculate area under the curve for oral drug administration.${{AUC}\quad t_{1}\quad {to}\quad t_{2}} = {{\frac{{C1} + {C2}}{2}\left( {t_{1} - t_{2}} \right)\quad {AUC}\quad t_{last}\quad {to}\quad t_{\infty}} = \frac{Clast}{k}}$

[0050] First order process:$k = {{\frac{{LnC}_{1} - {L\quad n\quad C_{2}}}{{t1} - {t2}}k} = {0.0164\quad {hr}^{- 1}}}$

[0051] AUC from 0 to ∞ (PO)=98.2 μg/ml/hr

[0052] Concentrations at 72 hr and 96 hr were not included incalculation following IV administration.

[0053] Area Under the Curve (AUC from 0 to ∞): Intercept-Slope MethodArea  Under  the  Curve  (AUC  from  0  to  ∞):  Intercept-Slope  Method${{AUC}\quad \left( {{AUC}\quad {from}\quad 0\quad {to}\quad \infty} \right)\quad ({IV})}:={{\frac{C_{0}}{k}\quad k} = {0.021\quad {hr}^{- 1}}}$AUC  (AUC  from  0  to  ∞)  (IV):  452.4  µg/ml/hr${F({Bioavailability})} = {{\frac{98.2}{457.4}\quad \frac{{IV}\quad {dose}\quad 1.5}{{PO}\quad {dose}\quad 2.5}F} = {{13\% {V\left( {{volume}\quad {of}\quad {distribution}} \right)}} = {{\frac{Dose}{Co}\quad \frac{1.5\quad g}{9.6\quad {µg}\text{/}{ml}}\quad V} = {0.35\quad L\text{/}{kg}}}}}$${{Cl}\quad \left( {{systemic}\quad {clearance}} \right)} = {{\frac{Dose}{{AUC}\quad 0\quad {to}\quad \infty}\quad \frac{1.5\quad g}{452.4\quad {µg}\text{/}{ml}\text{/}{hr}}{Cl}} = {55.26\quad {ml}\text{/}{\min.}}}$

[0054] Not detectable level of diclazuril in urine samples by both HPLCand TLC methods suggested that hepatic clearance is the majorelimination route for diclazuril in horses.

Cl_(H)=Q_(H).E_(H)

[0055] EH is lower than 0.3 so diclazuril is found to have low hepaticextraction ratio.

[0056] In yet another embodiment of the invention, the above-describedpreferred compositions can be utilized in methods of treatment ofanti-protozoal infections in animals other than EPM. For example, apresently preferred treatment regimen for treatment of Piroplasmosis inhorses comprises administering to a 1000 lb horse a composition forintravenous administration comprised of between about 50 mg to about1,500 mg, but especially between about 250 mg and about 1000 mg and mostpreferably about 500 mg (about 1.1 mg/kg) of diclazuril, toltrazuril,toltrazuril sulfone or sulfonotoltrazuril dissolved in a suitable volumeof DMSO, DMA or the like. Suitable amounts of solvent will vary frombetween about 2 ml to about 30 ml per unit dose depending upon thechoice of the anti-protozoal agent and the choice of solvent. Accordingto the methods set forth herein, the composition can given once per day(SID) for a duration of between about 1 and about 20 days but especiallybetween about 1 and about 10 days and most preferably about 4 days.Alternatively, a loading dose of the composition can be given to achieverapid critical plasma concentrations on day one of the regimen followedby a maintenance dose for a shorter duration of therapy, e.g., betweenabout 2 and about 10 days. In yet another embodiment, the compositionscan be specially formulated for sustained release as set forth belowsuch that a single administration of the anti-protozoal agent ispossible or, alternatively a single intraveneous loading dose followedby an intramuscular or subcutaneous dose for sustained critical bloodlevels.

[0057] As set forth above, clazuril, diclazuril, toltrazuril,toltrazuril sulfone and sulfonotoltrazuril as well as othertriazine-based anti-coccidials agents that are useful in the prophylaxisand therapy of equine protozoan myeloencephalitis (EPM) and otherprotozoal diseases in man and animals. These compounds are hydrophobicand highly water insoluble.

[0058] Prior to the teachings set forth herein, there had been nosuitable water soluble formulation for parenteral administration ofthese agents. Accordingly, the present invention provides compositionscomprised of a water-soluble form of at least one anti-protozoal agentwhich is useful for the treatment of a protozoal infection in man or inanimals and a method for making such a composition. In a presentlypreferred embodiment, the composition is comprised of the sodium salt ofone or more triazine-based anticoccidial agents, including, but notlimited to clazuril, diclazuril, letrazuril, toltrazuril, toltrazurilsulfone, sulfonotoltrazuril or mixtures thereof.

[0059] A presently preferred way to increase the water solubility ofsuch drugs is to make their salt forms. Clazuril, diclazuril,toltrazuril, toltrazuril sulphone and sulfonotoltrazuril are weak acidsbecause of the imide hydrogen present in all five compounds as shownbelow. The anion formed when this hydrogen gets abstracted is resonancestabilized. Thus, these acidic compounds can be reacted with a base toform the corresponding salt and water in a typical acid-base titrationreaction. For instance, the sodium salt of diclazuril was formed byreacting diclazuril with sodium hydroxide as set forth below in a 1:1molar ratio.

[0060] Evidence for salt formation:

[0061] 1. Molar ratio of sodium hydroxide required for compete titrationof diclazuril was 1:1.

[0062] 2. The salt dissolves immediately in water while diclazuril iscompletely insoluble in water.

[0063] 3. Diclazuril is a white powder. The sodium salt of diclazurilhas a brownish tan color.

[0064] 4. Mass spectral analysis in the negative mode of the sodium saltof diclazuril and diclazuril itself was conducted. The expectedmolecular weight of the molecular anion of the salt and the parentcompound is 404.971. The measured molecular weight matched exactly tothe expected weight (Data not shown).

[0065] Therefore, clazuril, diclazuril, toltrazuril, toltrazurilsulphone, and sulfonotoltrazuril, as well as certain othertriazine-based anticoccidials, have in common an acidic imide hydrogenwhich allows for an acid-base titration reaction with a base to give thecorresponding salts and water. This was demonstrated with the synthesisof the sodium salt diclazuril as set forth more fully in Example 5below. Give the teachings provided herein, it can be appreciated thatother alkali bases including, but not limited to Ca(OH)₂, KOH, LiOH canalso be used.

Example 5 Synthesis of a Water-Soluble Salt of Diclazuril

[0066] Diclazuril has a very low solubility in water (<1×10⁻⁷ at pH 6.5)and can in fact be considered as practically insoluble in water. Apresently preferred plasma drug concentration of about 8 μg/ml for about25-30 days is needed for the effective treatment of equine protozoanmyeloencephalitis (EPM). The oral bioavailability of diclazuril is verylow. Thus, a parenteral formulation is desired.

[0067] One of the methods to increase the water solubility of poorlywater-soluble drugs is to make water-soluble salts of the drug.Diclazuril can be considered as a weak acid because of the presence ofthe imide hydrogen as shown below:

[0068] The anion formed when this acidic hydrogen gets abstracted isstabilized by resonance below:

Resonance Structures of Diclazuril Imide Anion

[0069] Since diclazuril is a weak acid, it can react with a base to givea salt and water. NaOH was chosen as the presently preferred base and ahacid-base reaction was carried out wherein I gram of diclazuril wassuspended in 50 ml of ethanol in a 200 ml volumetric flask. To this,phenolphthalein indicator was added to indicate equivalence point oftitration. A buret was filled with 1M NaOH. The base was slowly addedfrom the buret to the solution. The titration was stopped when a changein the color of the phenolphthalein indicator (colorless to pink) wasobserved. At the equivalence point, it was observed that the white milkysuspension of diclazuril had changed to a clear pink solution. This wasthought to be due to the conversion of the acid to the salt. Thesolution was re-crystalized by evaporation in an oven set at 75° C. fortwo hours.

[0070] Calculation of moles of NaOH required to titrate 1 mole ofdiclazuril indicated that the molar ratio was 1: 1, suggesting acid-basereaction and thus salt formation.

[0071] While it is possibe that the nitrile and imide functional groupscan be hydrolyzed by a base. This probably does not occur, however,because hydrolysis of nitrites and imides requires more severeconditions including refluxing for several hours. In addition, thereaction was conducted in ethanol thereby eliminating water required forthe hydrolysis reaction.

[0072] Evidence for Salt Formation

[0073] 1. The titration experiment strongly suggests acid-base reaction.

[0074] 2. Mass spectral evidence: Fourier transform matrix-assistedlaser desorption ionization mass spectrometry (MALDI-FTMS) withdithranol matrix was used to measure the mass spectra of diclazurilstarting material and diclazuril free acid obtained from the sodium saltof diclazuril. The expected molecular weight of diclazuril anion(C₁₇H₈C₁₃N₄o₂—) is 404.972. The measured molecular weight is 404.971.Isotopic peaks arising from chlorine were measured at m/z of 406.967 and408.965. A cluster of peaks at molecular weight 333.971 in the spectrumcorresponds to a fragment peak of the molecular ion formed after theloss of two chlorine atoms. The sodium salt was converted to the freeacid by extraction with ethyl acetate and treatment with acetic acidprior to mass spectral measurement. Again, the expected molecular ion ofdiclazuril anion is 404.972 and the measured molecular weight of 404.971matched exactly to the expected weight. Isotopic peaks arising fromprimarily from chlorine were also measured. Cluster of peaks seen at333.971 again corresponds to a fragment peak obtained after the loss oftwo chlorine ions. An additional cluster of peaks at molecular weight395.952 that was not seen in the mass spectrum of diclazuril startingmaterial was seen in the mass spectrum of diclazuril free acid obtainedfrom the sodium salt. This peak is thought to be either an unidentifiedfragment peak of the molecular ion, peak arising from impurities orminor side product of the titration reaction. Further analysis by HPLCis warrented to identify whether this peak is a fragmentation product oran impurity.

[0075] pH Stability of Sodium Salt of Diclazuril

[0076] The manufacturer of diclazuril, JANSEEN Research Foundation,listed the pKa of diclazuril to be 5.92 in its physico-chemicalcharacteristics of diclazuril. It can be expected that at pH's above thepKa of the compound ( i.e. pH>6), aqueous solution of diclazuril wouldstay in solution. Preliminary studies were conducted to determine the pHat which sodium salt would precipitate.

[0077] Sodium salt of diclazuril was dissolved in water. It went intosolution almost immediately. The pH of the aqueous solution was 12.4. Tothis solution, 1N HCl was added dropwise while stirring and the pH atwhich the solution began to turn cloudy was recorded. This pH was foundto be 10.5. This would indicate that the pKa of diclazuril is not 5.92as indicated by JANSSEN but closer to 10.5. Actually, a check ofreference pKa's of organic compounds shows that imides have a pKa in therange of 8.3-9.6.

[0078] The result of the pH stability study indicated that aqueousformulation of sodium salt of diclazuril has to be formulated preferablyat a higher pH close to 11 to ensure that the drug stays in solution.This will not cause a problem as there are many drugs on the market thatare formulated at higher pHs for exactly the same reason.

[0079] Accordingly, the present invention provides a composition usefulfor the treatment of a protozoal infection in man or in animalscomprised of a water-soluble form of a triazine-based anticoccidialincluding, but not limited to, clazuril, diclazuril, toltrazuril,toltrazuril sulfone and sulfonotoltrazuril. In a presently preferredembodiment of the invention, the water soluable triazine-basedanticoccidial is a sodium salt of clazuril, diclazuril, toltrazuril,toltrazuril sulfone, sulfonotoltrazuril or a mixture thereof.

[0080] The preferred compositions can be utilized in methods oftreatment of anti-protozoal infections in man and in animals. Forexample, a presently preferred treatment regimen for treatment of EPMcomprises administering to a 1000 lb horse a composition for intraveneusadministration comprised of between about 50 mg to about 1,500 mg, butespecially between about 250 mg and about 1000 mg and most preferablyabout 500 mg (about 1.1 mg/kg) of a water-soluble sodium salt ofdiclazuril, toltrazuril, toltrazuril sulfone or sulfonotoltrazurildissolved in a suitable volume of water or stored in a freeze-driedpreparation for admixing with a suitable amount of sterile water forinjection at the time of utilization. Suitable amounts of water for thecomposition will vary from between about 2 ml to about 30 ml per unitdose depending upon the choice of the anti-protozoal agent and theparenteral route of administration.

[0081] According to the methods set forth herein for EPM treatment, thecomposition can given once per day (SID) for a duration of between about10 and about 35 days but especially between about 20 and about 30 daysand most preferably about 28 days. Alternatively, a loading dose of thecomposition can be given to achieve rapid critical plasma concentrationson day one of the regimen followed by a maintenance dose (see, Example 1above) for a shorter duration of therapy, e.g., between about 15 andabout 25 days.

[0082] It is also specifically contemplated that the water-soluble formsof the triazine-based anticoccidials provided herein can be utilized insuitable oral formulations for treatment and prevention of protozoalinfections and infestations in man and in animals. The solubility of thecompositions provided herein greatly enhance the oral bioavailability ofthe active anti-protozoal agent and accordingly lessen the dosagerequirement to roughly the equivalent of that of the parenteralformulations. Given the teachings provided herein, the skilled artisancan optimize the dosage requirements and the therapeutic regimen for aparticular formulation depending upon the condition being treated orprevented and the species of animal.

[0083] One embodiment of the present invention provides a compositionuseful for the treatment of a Cryptosporidium spp. infection especiallyin man or in animals comprised of a water-soluble form of atriazine-based anticoccidial including, but not limited to, clazuril,diclazuril, toltrazuril, toltrazuril sulfone or sulfonotoltrazuril. In apresently preferred embodiment of the invention, the water soluabletriazine-based anticoccidial is a sodium salt of clazuril, diclazuril,toltrazuril, toltrazuril sulfone, sulfonotoltrazuril or a mixturethereof and can be used parenterally or orally in a therapeutictreatment regimen. It is also contemplated that the above-describedcompositions comprised of a triazine-based anticiccidial and a suitablesolvent, e.g., DMSO or DMA, can be utilized parenterally to treatCryptosporidium spp. infection in man or in animals.

[0084] For example, a presently preferred treatment regimen fortreatment of Cryptosporidium spp. infection comprises administering toan approximately 200 lb human subject from between about 10 mg to about400 mg, but especially between about 25 mg and about 300 mg and mostpreferably about 200 mg (about 1.1 mg/kg) of a water-soluble sodium saltof diclazuril, toltrazuril, toltrazuril sulfone or sulfonotoltrazurildissolved in a suitable volume of water or stored in a freeze-driedpreparation for admixing with a suitable amount of sterile water forinjection at the time of utilization. Suitable amounts of water for thecomposition will vary from between about 2 ml to about 10 ml per unitdose depending upon the choice of the anti-protozoal agent and the routeof route of administration. It can certainly be appreciated that theoral formulations of the water-soluble salts of these agents can be in aliquid, semi-solid or solid form as a pill tablet, elixir and the like.

[0085] Current recommendations for treatment of EPM with pyrimethamineand a sulfonamide are set forth in the above-cited articles by Dr. ClaraFenger (1995) and Dr. Joe Bertone (1995 which are hereby incorporated byreference. Accordingly, in yet another embodiment of the invention, thecomposition is comprised of at least one anti-protozoal agent as setforth above and can also comprise a reduced amount of a sulfonamideand/or pyrimethamine for the parenteral, erg., the intranasalformulations.

[0086] One skilled in the art can appreciate that depending upon thecompound which is selected as the therapeutic agent, the method ofsolubilization of the compound for inclusion in the composition providedherein can vary and can be readily ascertained based upon known chemicalproperties of the selected compound, as found, e.g., in the materialsafety data sheets (MSDS) for the particular compound or via knownmethods of synthesis etc. In particular, methods for solubilization ofthe active compound and formulation of compositions that are speciallyadapted for intranasal administration are known as set forth, e.g., inU.S. Pat. Nos.: 4,284,648, 4,428,883; 4,315,925 and 4,383,993, thecontents of which are hereby incorporated by reference.

[0087] In view of the mixed results of the cases of EPM treated to dateby the oral route, it is very surprising that by utilizing a differentroute of administration (i.e., intranasal) and decreasing the dose, thatone could achieve better results, especially when using ananti-coccidial agent which has to exert its effect on the organism inthe horse. Furthermore, while the solubilization techniques suggestedabove were known, it has not been heretofore suggested that such can beused for solubilizing triazine-based anti-coccidial agents, such asdiclazuril, toltrazuril, and toltrazuril sulfone, in order to providecompositions for intranasal administration. The intranasal route allowsfor direct absorption of drug through the nasal mucosa and directlyacross the blood brain barrier into the CSF without first having to passthrough the liver. This may produce the unexpectedly better result seenwith this route of administration, even with drastically lower unitdosages of drug.

[0088] For example, one embodiment of the invention utilizes acomposition for treatment of EPM adapted for intranasal administrationwhich is comprised of toltrazuril, toltrazuril sulfone orsulfonotoltrazuril in a therapeutically effective amount in which theunit dose is less than the unit dose required for a therapeuticallyamount of toltrazuril, toltrazuril sulfone or sulfonotoltrazuril as isnormally required for oral administration of either drug. In preferredembodiments, the therapeutically effective amount of the compositionscomprised of toltrazuril, toltrazuril sulfone or sulfonotoltrazuril isbetween about 1/4th and 1/100th but especially about 1/10th of thetherapeutically effective amount of toltrazuril, toltrazuril sulfone orsulfonotoltrazuril that is required for oral administration. Currentlyrecommended oral dosage levels of toltrazuril, toltrazuril sulfone orsulfonotoltrazuril are 5-10 mg/kg per unit dose administered once dailyorally such that a typical 500 kg horse would receive between about 2.5to about 5 grams of active ingredient per day.

[0089] Thus, one presently preferred embodiment of the inventioncomprises a composition comprised of toltrazuril, toltrazuril sulfone orsulfonotoltrazuril in solution with N-methyl-glucamine and water. Forinstance, a typical example of how to solubilize toltrazuril for use inthe compositions contemplated by the invention is to combine about 10 mgof toltrazuril with about 100 mg of N-methyl glucamine and about 10 ccof water. One can appreciate given the known properties of toltrazuril,toltrazuril sulfone or sulfonotoltrazuril that other solvents can beutilized for use in preparation of the above compositions.

[0090] In one embodiment of the invention, the effective amount oftoltrazuril, toltrazuril sulfone or sulfonotoltrazuril per unit dose forintranasal administration is between about 50 mg and about 1,500 mg, butespecially between about 100 mg and about 750 mg. In one embodiment, theeffective amount of toltrazuril, toltrazuril sulfone orsulfonotoltrazuril per unit dose is about 500 mg. Depending upon thedosage required for either treatment or prevention of EPM (which can beoptimized utilizing methods known in the art) the appropriate amount oftoltrazuril or sulfonotoltrazuril can be solubilized for production of aunit dose or the unit doses can be combined in a multiple treatment vialor container.

[0091] Likewise, another embodiment of the invention provides acomposition adapted for intranasal administration which is comprised ofdiclazuril in a therapeutically effective amount in which the unit doseis less than the unit dose for a therapeutically amount of diclazurilthat is required for oral administration to treat EPM. In preferredembodiments, the therapeutically effective amount of the compositionscomprised of diclazuril is between about 1/4th and 1/100th butespecially about 1/10th of the therapeutically amount of diclazuril thatis required for oral administration.

[0092] A typical example of the solubilization of diclazuril for use inthe compositions contemplated by the invention is to combine about 10 mgof diclazuril with about 20 mg of nicotinamide, about 300 mg ofpropylene glycol and about 9-10 cc of water. It is contemplated thatother solvents can be utilized for preparation of the compositionscomprising diclazuril. The skilled artisan can select from among knownsolvents to fit a particular embodiment, see, e.g. Janssen PharmaceuticaSafety Information for Diclazuril (R-64433) the contents of which isincorporated herein by reference.

[0093] In one embodiment of the invention, the effective amount ofdiclazuril per unit dose for intranasal administration is between about50 mg and about 1,000 mg, but especially between about 100 mg and about750 mg. In one embodiment, the effective amount of diclazuril per unitdose is about 350 mg. Depending upon the dosage required for eithertreatment or prevention of EPM (which can be optimized utilizing methodsknown in the art), the appropriate amount of diclazuril can thus besolubilized for production of a unit dose or the unit doses can becombined in a multiple treatment vial or container.

[0094] Accordingly, therefore, another embodiment of the inventionprovides a composition adapted for intranasal administration which iscomprised of diclazuril, and DMSO in a therapeutically effective amountin which the unit dose is less than the unit dose for a therapeuticallyamount of diclazuril that is required for oral administration to treatEPM. In preferred embodiments, the therapeutically effective amount ofthe compositions comprised of diclazuril and DMSO is between about 1/4thand 1/100th but especially about 1/10th of the therapeutically amount ofdiclazuril that is required for oral administration One presentlypreferred embodiment of the invention, comprises a composition adaptedfor intranasal administration comprising diclazuril solubilized in DMSO.The diclazuril is placed into solution in the DMSO and can be furtherformulated with other pharmaceutically acceptable carriers andexcipients for intranasal administration to fit a particular treatmentregimen. A typical unit dosage for administration is between about 50 mgand about 750 mg but is especially between about 100 mg and 500 mg andpreferably about 250 mg. In one example, 500 mg of diclazuril isdissolved in 10-15 cc of DMSO and can be utilized for intra nasaladministration. This same composition can also be utilized forintravenous administration for treatment of EPM by methods provided forherein.

[0095] Typically, the subject equid can be treated via intranasaladministration of the solution on a daily basis utilizing, e.g., acatheter and syringe to apply the solution directly to the intranasalmucosa. Depending upon the disposition of the animal, proper restraintshould be utilized such as a nose twitch before initiation of theprocedure. After proper restraint of the animal, the catheter isadvanced up the animal's nostril to the level of the oropharnyx. Most ofthe solution is injected through the catheter onto the nasal mucosa atthe oropharnyx with the remainder being injected as the catheter isslowly withdrawn. For daily administration, as with some of the methodsof treatment provided herein, the nostrils may be alternated so as tominimize irritation of the mucosa. The total volume of the compositionapplied intranasally typically will not exceed 15-20cc peradministration and preferably does not exceed about 10 cc peradministration.

[0096] Additionally, as set forth in greater detail below, the abovesolution comprised of diclazuril and DMSO can be further adapted forsustained release of the diclazuril by any of a number of methods knownin the art, including the use of excipients and controlled releasedelivery systems comprised of, e.g., sucrose acetete isobutyrate, methylcellulose, or microparticles and the like. Frequency of dosing and unitdosage amounts for the sustained compositions set forth herein willvary, of course, depending upon the release characteristics of thedelivery system and the specific formulation. However, given theteachings of the invention, optimization of dosage and the therapeuticregimen will be routine to the skilled artisan.

[0097] Yet another embodiment of the invention comprises a compositionfor treatment of EPM adapted for intranasal administration which iscomprised of the anti-protozoal nitazoxanide in a therapeuticallyeffective amount in which the unit dose is less than the unit doserequired for a therapeutically effective amount of nitazoxanide (as setforth in the references cited above) as would normally be required forthe oral administration thereof. In preferred embodiments, thetherapeutically effective amount of the compositions comprised ofnitazoxanide is between about 1/4th and 1/100th but especially about1/10th of the therapeutically effective amount of nitazoxanide thatwould be required for oral administration.

[0098] It can be appreciated that other nitrothiazoles theirderivatives, analogs, isomers, salts, and natural metabolites can alsobe utilized in the compositions for treatment and prevention of EPMdisclosed herein.

[0099] The dosage of nitazoxanide can vary from between about 100 mg toabout 1,500 mg depending upon the specific formulation, but isespecially between about 250 mg and about 1000 mg for the intranasalformulations.

[0100] Another embodiment of the invention provides a compositionadapted for equine intranasal or other parenteral administration whichis comprised of pyrimethamine and at least one sulfonamide in atherapeutically effective amount in which the unit dose is less than theunit dose for a therapeutically amount of the same drug combination thatis required for oral or other parenteral administration to treat EPM. Inpreferred embodiments, the therapeutically effective amount of thecompositions comprised of pyrimethamine and the sulfonamide is betweenabout 1/4th and 1/100th but especially about 1/10th of thetherapeutically amount of the same drug combination that is required fororal or other parenteral administration. These compositions can, ofcourse, contain more than one sulfonamide in combination withpyrimethamine, as well as additional pharmaceutically acceptableexcipients and adjuvants.

[0101] Also contemplated are compositions adapted for parenteral (e.g.,intramuscular, subcutaneous or intravenous) administration which arecomprised of at least one chemical agent one which has anti-protozoalactivity, e.g., a triazine based anticoccidial (a triazinedione or atriazinetrione e.g., diclazuril, toltrazuril or sulfonotoltrazuril ) ora nitrothiazole derivative, in a therapeutically effective amount inwhich the unit dose is less than the unit dose for a therapeuticallyeffective amount of the chemical agent that is required for oraladministration to treat or prevent EPM. In preferred embodiments, thetherapeutically effective amount of the compositions comprised of theanti-protozoal is between about 1/2th and 1/100th but especially about1/10th of the therapeutically amount of compound that is required fororal administration.

[0102] The invention also provides methods for treating and preventingEPM in an equine, e.g., a horse, which comprise the intranasaladministration of a composition such as those provided by the invention.The optimization of the unit dosage amounts and the treatment regimencan be accomplished utilizing methods which are generally known to theskilled artisan.

[0103] The invention further provides methods for treating andpreventing EPM in an equine, e.g., a horse, which comprise theparenteral, i.e., subcutaneous, intramuscular, intravenous, ortransdermal administration of a composition such as those provided bythe invention. The optimization of the unit dosage amounts and thetreatment regimen can be accomplished utilizing methods which aregenerally known to the skilled artisan.

[0104] In another embodiment of the invention, any of the antiprotozoalagents disclosed herein but especially the triazine-based anticoccidialcompounds or the nitrothiazoles including, but not limited todiclazuril, toltrazuril or toltrazuril sulfone, sulfonotoltrazuril andnitazoxanide can be placed in a composition adapted for sustainedrelease. The sustained release composition can comprise any of a numberof controlled delivery systems such as microparticles (microspheres ormicrocapsules), gels, and the like for formulated for injection orabsorption. The sustained release compositions of the invention can beadministered by any oral or parenteral route including intramuscular,subcutaneous, or intravenous injection. Likewise, the sustained releasecomposition can be adapted for transmucosal or transdermal delivery viaa patch, topical application, intranasal or intrauterine delivery andthe like.

[0105] Materials useful for preparation of the microspheres ormicrocapsules (microparticles) can include any biocompatable andpreferably biodegradable polymer, copolymer or blend. Suitable polymersinclude polyhydroxyl acids, polyorthoesters, polylactones,polycarbonates, polyphosphazenes, polysaccharides, proteins,polyanhydrides, copolymers thereof and blends thereof. Suitablepoly(hydroxy acids) include polyglycolic acid (PGA), polylactic acid(PLA), and copolymers thereof. Preferably the microparticles includepoly(D,L-lactic acid) and/or poly(D, L-lactic-co-glycolic acid).Particles with degredation and release times ranging from days to weeksor months can be designed and fabricated, based on factors such as thematerials used to prepare the microparticles. Of course, the sustainedrelease compositions contemplated can be utilized in methods oftreatment or for prevention of EPM.

[0106] The microparticles can be prepared using may method which doesnot destroy the activity of the active compound. Microparticles can beprepared using single and double emulsion solvent evaporation, spraydrying, solvent extraction, solvent evaporation, phase separation,simple and complex coacervation, interfacial polymerization and othermethods well known to those of ordinary skill in the art.

[0107] Methods developed for making microspheres for drug delivery aredescribed in the literature, for example, as described in Doubrow, M.,Ed, Microcapsules and Nanoparticles in Medicine and Pharmacy, CRC Press,Boca Raton, 1992. See also, U.S. Pat. Nos. 5,407,609 and 5,654,008 theteachings of which are incorporated herein by reference for methods ofmaking microspheres.

[0108] One specific example of a preferred embodiment of a controlleddelivery system of the invention is a composition comprised of any ofthe afore-mentioned therapeutic agents, (including the any of thetriazine-based anticoccidials and/or nitazoxanide) and sucrose acetateisobutyrate (SAIB) as set forth is U.S. Pat. No. 5,747,058, the contentsof which are incorporated herein by reference. The compositionscomprised of an anti-protozaol agent and SAIB, depending upon theformulation, can be administered topically, (e.g., transdermally ortransmucosally), subcutaneously or intramuscularily.

[0109] Another specific example of a controlled release formulationembodied by the invention is a composition comprised of any of theafore-mentioned therapeutic agents, (including the any of thetriazine-based antioccidials and/or nitazoxanide) and methylcelulose(e.g., Methocel). It can be appreciated that other controlled releaseformulations can be used including other degradable or non-degradableexcipients, although degradable excipients are preferred. Thecompositions comprised of an anti-protozaol agent and Methocel,depending upon the formulation, can be administered topically, (e.g.,transdermally or transmucosally), subcutaneously or intramuscularily.

[0110] Furthermore, the invention provides the above compositions foruse in methods of treating and preventing another common problem ofthoroughbred racehorses, a condition called laryngeal hemiplegia, whichhas an unknown etiology. While not wanted to be limited by theory, it isbelieved that laryngeal hemiplegia may be caused by or exacerbated byEPM. Laryngeal hemiplegia is a paralysis of the abductor muscles thatopen the arytenoid cartilages of the throat due to what is believed tobe recurrent laryngeal nerve damage. The arytenoid cartilage cannotabduct to open the airway and causes the affected horse to make a“roaring” noise when exercised and he breathes heavily. Therefore,laryngeal hemiplegia, and other potentially EPM-associated diseases andconditions can be treated or prevented by the present invention.

[0111] Additional objects, advantages and other novel features of theinvention will be set forth in part in the description that follows andin part will become apparent to those skilled in the art uponexamination of the foregoing or may be learned with the practice of theinvention.

[0112] The foregoing description of a preferred embodiment of theinvention has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit theinvention to the precise form disclosed. Obvious modifications orvariations are possible in light of the above teachings. The embodimentwas chosen and described to provide the best illustration of theprinciples of the invention and its practical application to therebyenable one of ordinary skill in the art to utilize the invention invarious embodiments and with various modifications as are suited to theparticular use contemplated. All such modifications and variations arewithin the scope of the invention as determined by the appended claimswhen interpreted in accordance with the breadth to which they arefairly, legally and equitably entitled.

What is claimed is:
 1. A composition useful for the treatment andprevention of protozoan infections in man and in animals that is adaptedfor parenteral administration, comprising at least one anti-protozoaldrug.
 2. The composition of claim 1, wherein the anti-protozoal drug isa triazine-based anticoccidial.
 3. The composition of claim 2, whereinthe triazine-based anticoccidial is selected from the group consistingof clazuril, diclazuril, letrazuril, toltrazuril and mixtures thereof.4. The composition of claim 2, wherein the triazine-based anticoccidialis sulfonotoltrazuril.
 5. The composition of claim 2, wherein thetriazine-based anticoccidial is toltrazuril sulfone.
 6. The compositionof claim 2, further comprising a suitable solvent of the triazine-basedanticocidial.
 7. The composition of claim 6, wherein the solvent isselected from the group consisting of DMSO, DMA and water.
 8. Thecomposition of claim 6, wherein the triazine-based anticoccidial isselected from the group consisting of clazuril, diclazuril, letrazuril,toltrazuril and mixtures thereof and the solvent is DMSO.
 9. Thecomposition of claim 6, wherein the triazine-based anticoccidial isdiclazuril sulffone and the solvent is DMSO.
 10. The composition ofclaim 6, wherein the triazine-based anticoccidial is sulfonotoltrazuriland the solvent is DMSO.
 11. The composition of claim 6, wherein thetriazine-based anticoccidial is toltrazuril sulfone and the solvent isDMSO.
 12. The composition of claim 6, wherein the triazine-basedanticoccidial is water-soluble and the solvent is water.
 13. Thecomposition of claim 2, wherein the triazine-based anticoccidial is awater-soluble salt of a triazine-based anticoccidial selected from thegroup consisting of clazuril, diclazuril, letrazuril, toltrazuril andmixtures thereof.
 14. The composition of claim 13, wherein thetriazine-based anticoccidial is a sodium salt of a triazine-basedanticoccidial selected from the group consisting of clazuril,diclazuril, letrazuril, toltrazuril and mixtures thereof.
 15. Thecomposition of claim 2, wherein the triazine-based anticoccidial is awater-soluble salt of toltrazuril sulfone.
 16. The composition of claim15, wherein the triazine-based anticoccidial is a sodium salt oftoltrazuril sulfone.
 17. The composition of claim 2, wherein thetriazine-based anticoccidial is a water-soluble salt ofsulfonotoltrazuril.
 18. The composition of claim 17, wherein thetriazine-based anticoccidial is a sodium salt of sulfonotoltrazuril. 19.The composition of claim 2, wherein the triazine-based anticoccidial isa water-soluble salt of diclazuril.
 20. The composition of claim 19,wherein the triazine-based anticoccidial is a sodium salt of diclazuril.21. The composition of claim 1, wherein the composition is formulatedfor a parenteral administration selected from the group consisting ofintraveneous, intramuscular, subcutaneous, intranasal, transdermal andtransmucosal.
 22. The composition of claim 2, fruther comprising a drugselected from the group consisting of a sulfonamide, pyrimethamine,nitazoxanide, a non-steroidal anti-inflammatory agent, and mixturesthereof.
 23. A composition useful for the treatment and prevention ofprotozoan infections in man and in animals that is adapted for oraladministration, comprising a water-soluble salt of a triazine-basedanticoccidial.
 24. The composition of claim 23, wherein thetriazine-based anticoccidial is selected from the group consisting ofclazuril, diclazuril, letrazuril, toltrazuril and mixtures thereof. 25.The composition of claim 23, wherein the triazine-based anticoccidial issulfonotoltrazuril.
 26. The composition of claim 23, wherein thetriazine-based anticoccidial is toltrazuril sulfone.
 27. The compositionof claim 23 wherein the water-soluble salt is a sodium salt of thetriazine-based anticoccidial.
 28. The composition of claim 23, whereinthe composition is adapted for oral administration such that the watersoluable salt of the triazine-based anticoccidial is protected fromprecipitation in the acidic environment of the stomach.
 29. Thecomposition of claim 28, wherein the composition formulated as a pill orcapsule and said pill or capsule is enteric coated to protect saidcomposition from the acidic pH of the stomach.
 30. A method of treatinga protozoal infection in man or in animals, comprising the parenteraladministeration of a therapeutically effective amount of a compositioncomprised of at least one triazine-based anticoccidial drug.
 31. Themethod of claim 30, wherein the triazine-based anticoccidial is selectedfrom the group consisting of clazuril, diclazuril, letrazuril,toltrazuril and mixtures thereof.
 32. The method of claim 30, whereinthe triazine-based anticoccidial is diclazuril.
 33. The method of claim30, wherein the triazine-based anticoccidial is sulfonotoltrazuril. 34.The method of claim 30, wherein the triazine-based anticoccidial istoltrazuril sulfone.
 35. The method of claim 30, wherein thetriazine-based anticoccidial is a water-soluble salt of a triazine-basedanticoccidial.
 36. The method of claim 35, wherein the water-solublesalt of the triazine based compound is a sodium salt of atrtiazine-based antioccidial selected from the group consisting ofclazuril, diclazuril, letrazuril, toltrazuril and mixtures thereof. 37.The method of claim 30, wherein the triazine-based anticoccidial is awater-soluble salt of a sulfonotoltrazuril.
 38. The method of claim 30,wherein the triazine-based anticoccidial is a water-soluble salt oftoltrazuril sulfone.
 39. The method of claim 30, wherein the parenteraladministration route is selected from the group consisting ofintraveneous, intramuscular, subcutaneous, intranasal, transdermal andtransmucosal.
 40. The method of claim 30, wherein the compositionfurther comprises a suitable solvent of the triazine-based anticocidial.41. The method of claim 30, wherein the protozoal infection is selectedfrom the group consisting of equine protozoal myeloencephalitis, equinepiroplasmosis, and human cryptosporidiosis.
 42. The method of claim 30wherein the therapeutically effective amount of the triazine-basedanticoccidial is from between about 0.01 mg/kg and about 20 mg/kg. 43.The method of claim 40, wherein the protozoal infection is equineprotozoal myeloencephalitis in an equid, the triazine basedanticoccidial is diclazuril, the solvent is DMSO, the parenteral routeis intraveneous and the therapeutically effective amount is from betweenabout 0.01 mg/kg and about 20 mg/kg.
 44. The method of claim 43 whereinthe therapeutically effective amount is from between about 1 mg/kg andabout 10 mg/kg.
 45. The method of claim 43 wherein the therapeuticallyeffective amount is from between about 3 mg/kg and about 6 mg/kg. 46.The method of claim 40, wherein the protozoal infection is equineprotozoal myeloencephalitis in an equid, the triazine basedanticoccidial is sulfonotoltrazuril, the solvent is DMSO, the parenteralroute is intraveneous and the therapeutically effective amount is frombetween about 0.01 mg/kg and about 20 mg/kg.
 47. The method of claim 46wherein the therapeutically effective amount is from between about 1mg/kg and about 10 mg/kg.
 48. The method of claim 46 wherein thetherapeutically effective amount is from between about 3 mg/kg and about6 mg/kg.
 49. The method of claim 40, wherein the protozoal infection isequine piroplasmosis in an equid, the triazine based anticoccidial issulfonotoltrzuril, the solvent is DMSO, the parenteral route isintraveneous and the therapeutically effective amount is from betweenabout 0.01 mg/kg and about 20 mg/kg.
 50. The method of claim 49 whereinthe therapeutically effective amount is from between about 1 mg/kg andabout 10 mg/kg.
 51. The method of claim 49 wherein the therapeuticallyeffective amount is from between about 3 mg/kg and about 6 mg/kg. 52.The method of claim 40, wherein the protozoal infection is equinepiroplasmosis in an equid, the triazine based anticoccidial isdiclazuril, the solvent is DMSO, the parenteral route is intraveneousand the therapeutically effective amount is from between about 0.01mg/kg and about 20 mg/kg.
 53. The method of claim 52 wherein thetherapeutically effective amount is from between about 1 mg/kg and about10 mg/kg.
 54. The method of claim 52 wherein the therapeuticallyeffective amount is from between about 3 mg/kg and about 6 mg/kg. 55.The method of claim 40, wherein the protozoal infection is humancryptosporidiosis in a human subject, the parenteral route isintraveneous and the therapeutically effective amount is from betweenabout 0.01 mg/kg and about 20 mg/kg.
 56. The method of claim 52 whereinthe therapeutically effective amount is from between about 1 mg/kg andabout 10 mg/kg.
 57. The method of claim 52 wherein the therapeuticallyeffective amount is from between about 3 mg/kg and about 6 mg/kg. 58.The method of claim 30, wherein the triazine based anticoccidial is awater -soluble salt of a triazine-based anticoccidial selected from thegroup consisting of clazuril, diclazuril, letrazuril, toltrazuril,sulfonotoltrazuril and mixtures thereof, and the therapeuticallyeffective amount is from between about 0.01 mg/kg and about 20 mg/kg.59. The method of claim 55 wherein the therapeutically effective amountis from between about 1 mg/kg and about 10 mg/kg.
 60. The method ofclaim 55 wherein the therapeutically effective amount is from betweenabout 3 mg/kg and about 6 mg/kg.